Method and apparatus for analyzing individual cells or particulates using fluorescent quenching and/or bleaching

ABSTRACT

A method for analyzing a blood sample is provided that includes the steps of: providing a blood sample having one or more of each first and second constituents; admixing a colorant with the sample, which colorant is operative to cause the first constituents and second constituents to fluoresce and absorb light; illuminating at least a portion of the sample; e) imaging a portion of the sample; determining a fluorescence value for each the first constituents and second constituents; determining an optical density value for each of the first constituents and second constituents; and identifying the first constituents and the second constituents using the determined fluorescence and optical density values.

The present application is a continuation of U.S. patent applicationSer. No. 14/673,294 filed Mar. 30, 2015, which is a continuation of U.S.patent application Ser. No. 13/959,294 filed Aug. 5, 2013, which is acontinuation of U.S. patent application Ser. No. 13/645,075 filed Oct.14, 2012, which is a continuation of U.S. patent application Ser. No.13/330,236 filed Dec. 19, 2011, which is a divisional of U.S. Pat. No.8,081,303 filed Dec. 20, 2011, which is entitled to the benefit of andincorporates by reference essential subject matter disclosed in U.S.Provisional Patent Application Ser. No. 61/038,578, filed Mar. 21, 2008.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to apparatus and methods for analysis ofblood samples in general, and apparatus and methods for detecting,identifying, and enumerating constituents, such as cells orparticulates, within the sample in particular.

2. Background Information

Physicians, veterinarians and scientists have examined human andanimals' biologic fluids, especially blood, in order to determineconstituent quantities as well as to identify the presence of unusualconstituents not seen in healthy subjects. The constituents generallymeasured, quantified and identified include red blood cells (RBCs),white blood cells (WBCs), and platelets. RBC analyses can includedeterminations of RBC number, size, volume, shape, hemoglobin contentand concentration, and the hematocrit (also referred to as the packedcell volume). RBC analyses can also involve determining the presenceand/or concentration of certain components within the red cells such asDNA, RNA, including the detection of the presence and/or enumeration ofhematoparasites (e.g., malarial parasites) either in the RBCs ortrypanosomes which are extracellular or leishmaniasis organisms whichare in the WBCs as well as many other hematoparasites. WBC analyses caninclude a determination of the population frequency of WBC sub-typesgenerally referred to as a differential WBC count, as well as thenotification of any unusual cell types not found in healthy subjects.Platelet (or in certain animals including birds, reptiles and fish,thrombocytes which are similar in function to platelets in mammals butare about ten times larger and nucleated) analyses can include plateletnumber, size, shape texture, and volume determinations, includingdetermining the presence of clumps of platelets or thrombocytes withinthe sample.

Known blood examination techniques, described in detail medical textssuch as Wintrobe's Clinical Hematology 12^(th) Edition, generallydivides the examination methods into manual, centrifugal, and impedancetype methods. Manual methods typically involve the creation of anaccurately determined volume of a blood or fluid sample that isquantitatively diluted and visually counted in a counting chamber.Manual examination methods include examining a peripheral smear wherethe relative amounts of the particulate types are determined by visualinspection. Centrifugal examination methods involve centrifuging thesample, causing the sample to separate into constituent layers accordingto the relative densities of the constituents. The component layers canbe stained to enhance visibility or detection. Impedance methods involvethe examination of an accurate volume of blood which is treatedaccording to the particulate being measured; e.g., lysing RBCs forenumeration of the nucleated cells and volumetrically diluting thesample in a conductive fluid. The process typically involves monitoringa current or voltage applied to sample passing through a narrow passageto determine the effect particles have on the current/voltage as theparticles pass through in single file. Other techniques involveanalyzing the intensity and angle of scatter of light incident toparticulates passing single file through a light beam. Flow cytometricmethods can also be used that involve staining particulates of interestin suspension with fluorophores attached to antibodies directed againstsurface epitopes present on cell or particle types, exciting the stainedparticulates with light of appropriate wavelengths, and analyzing theemission of the individual particulates/cells.

All of the aforementioned methods, other than the peripheral smear orcentrifugal separation, require dispensing a precise volume of sample.Inaccuracies in the sample volume will result in quantitative errors ofthe same magnitude in the associated analysis. With the exception ofcentrifugal methods, all of the aforementioned methods also require thesample to be mixed with one or more liquid reagents or diluents, andalso require calibration of the instrument to obtain accurate results.In the case of peripheral smears, a high degree of training is needed toproperly examine the smear. A number of the aforementioned methodsgenerate large volumes of contaminated waste which is expensive tohandle. Additionally, the above-described methods are not suitable todetermine the complete blood count (CBC) in birds, reptiles, fish wherethe red blood cells and thrombocytes are nucleated and in certainmammals where the red blood cells size is very small and may be confusedwith platelets.

The amount of information that can be determined by examining the bloodof a human or animal is vast. It is particularly useful to determine theindices of RBCs; e.g., individual cell size, individual cell hemoglobincontent and concentration, and population statistics of RBCs within asample. The mean and dispersion statistics (e.g., coefficients ofvariation) for each of the aforementioned parameters can also provideimportant information, as is evidenced by their discussion within theabove-referenced text by Wintrobe, which has enabled physicians tobetter categorize disorders of RBCs.

SUMMARY OF THE INVENTION

A method and apparatus is provided for analyzing constituents within aquiescent blood sample. According to one aspect of the invention, amethod for analyzing a blood sample is provided that includes the stepsof: a) providing a substantially undiluted blood sample having one ormore first constituents and one or more second constituents, whichsecond constituents are different from the first constituents; b)depositing the sample into an analysis chamber adapted to quiescentlyhold the sample for analysis, the chamber defined by a first panel and asecond panel, both of which panels are transparent; c) admixing acolorant with the sample, which colorant is operative to cause the firstconstituents and second constituents to fluoresce upon exposure topredetermined first wavelengths of light, and which colorant isoperative to absorb light at one or more predetermined secondwavelengths of light; d) illuminating at least a portion of the samplecontaining the first constituents and the second constituents at thefirst wavelengths and at the second wavelengths; e) imaging the at leasta portion of the sample, including producing image signals indicative offluorescent emissions from the first constituents and the secondconstituents and the optical density of the first constituents and thesecond constituents; f) determining a fluorescence value for each thefirst constituents and second constituents using the image signals; g)determining an optical density value for each of the first constituentsand second constituents, which optical density is a function of thecolorant absorbed by the constituents, using the image signals; and h)identifying the first constituents and the second constituents using thedetermined fluorescence and optical density values.

According to another aspect of the present invention, a method foranalyzing a blood sample is provided that includes the steps of: a)providing a substantially undiluted blood sample having one or morefirst particulates and one or more second particulates, which secondparticulates are different from the first particulates; b) depositingthe sample into an analysis chamber adapted to quiescently hold thesample for analysis, the chamber defined by a first panel and a secondpanel, both of which panels are transparent; c) admixing a colorant withthe sample, which colorant is operative to cause the first particulatesand second particulates to fluoresce upon exposure to predeterminedfirst wavelengths of light, and which colorant is operative to absorblight at one or more predetermined second wavelengths of light; d)illuminating at least a portion of the sample containing the firstparticulates and the second particulates at the first wavelengths and atthe second wavelengths; e) imaging the at least a portion of the sample,including producing image signals indicative of fluorescent emissionsfrom the first particulates and the second particulates and the opticaldensity of the first particulates and the second particulates; f)determining one or more fluorescent emission values for each the firstparticulates and second particulates using the image signals; g)determining one or more optical density values for each of the firstparticulates and second particulates, which optical density is afunction of the colorant absorbed by the particulates, using the imagesignals; and h) identifying the first particulates and the secondparticulates using the determined fluorescent emission and opticaldensity values.

According to a still further aspect of the present invention, a methodfor analyzing a blood sample is provided that includes the steps of: a)providing a substantially undiluted blood sample having one or morefirst constituents and one or more second constituents, which secondconstituents are different from the first constituents; b) depositingthe sample into an analysis chamber adapted to quiescently hold thesample for analysis, the chamber defined by a first panel and a secondpanel, both of which panels are transparent; c) admixing a colorant withthe sample, which colorant is operative to cause the first constituentsand second constituents to fluoresce upon exposure to predeterminedwavelengths of light; d) illuminating at least a portion of the samplecontaining the first constituents and the second constituents at thewavelengths of light in a constant manner for a period of time; e)imaging the at least a portion of the sample at discrete points in timeduring the period, and producing image signals indicative of fluorescentemissions from the first constituents and the second constituents foreach discrete point in time; f) determining one or more fluorescentemission values for each of the first constituents and secondconstituents quiescently disposed within the sample using the imagesignals for each discrete point in time, and a rate of change for thefluorescent emission values between the discrete point in time for eachof the first and second constituents; and g) identifying the firstconstituents and the second constituents using the determined rate ofchange of the fluorescent emission values for each of the first andsecond constituents.

An advantage of the present invention is that it can be used todetermine characteristics of a blood sample using an extremely smallsample volume that may be obtained directly from the patient bycapillary puncture rendering it more useful for point of careapplication or from a venous sample if desired.

Another advantage of the present method is that it operates free ofexternal and internal fluidics, and independent of gravity ororientation, and therefore is adaptable for use in a hand held deviceand in microgravity conditions.

The present method and advantages associated therewith will become morereadily apparent in view of the detailed description provided below,including the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 are cross-sectional diagrammatic representations of analysischambers that may be used in the present method.

FIG. 5 is a diagrammatic planar view of a tape having a plurality ofanalysis chambers.

FIG. 6 is a diagrammatic planar view of a disposable container having ananalysis chamber.

FIG. 7 is a diagrammatic cross-sectional view of a disposable containerhaving an analysis chamber.

FIG. 8 is a diagrammatic schematic of an analysis device that may beused with the present method.

FIG. 9 is a graph diagrammatically illustrating an amount of fluorescentdecay as a function of time for neutrophils.

FIG. 10 is a graph diagrammatically illustrating an amount offluorescent decay as a function of time for lymphocytes.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present method utilizes an analysis chamber that is operable toquiescently hold a sample of substantially undiluted anticoagulatedwhole blood for analysis. The chamber is typically sized to hold about0.2 to 1.0 μl of sample, but the chamber is not limited to anyparticular volume capacity, and the capacity can vary to suit theanalysis application. The phrase “substantially undiluted” as usedherein describes a blood sample which is either not diluted at all orhas not been diluted purposefully, but has had some reagents addedthereto for purposes of the analysis. To the extent the addition of thereagents dilutes the sample, if at all, such dilution has no clinicallysignificant impact on the analysis performed. Typically, the onlyreagents that will be used in performing the present method areanticoagulants (e.g., EDTA, heparin) and colorants. These reagents aregenerally added in dried form and are not intended to dilute the sample.Under certain circumstances (e.g., very rapid analysis), it may not benecessary to add an anticoagulating agent, but it is preferable to do soin most cases to ensure the sample is in a form acceptable for analysis.The term “quiescent” is used to describe that the sample is depositedwithin the chamber for analysis, and the sample is not purposefullymoved relative to the chamber during the analysis; i.e., the sampleresides quiescently within the chamber. To the extent that motion ispresent within the blood sample, it will predominantly be that due toBrownian motion of the blood sample's foamed constituents, which motionis not disabling of the use of the device of this invention.

The colorant (e.g., a dye, stain, etc.), which is admixed with at leasta portion of the blood sample, facilitates quantitative analysis of theconstituents (e.g., WBCs and other nuclear containing cells, andparticulates including platelets, and other constituents containing DNAand/or RNA—e.g., intracellular or extracellular hematoparasites—etc.)that absorb the colorant. The cells and particulates may be collectivelyreferred to herein as “constituents” within the sample. The colorantfluoresces along characteristic wavelengths (e.g., 530 nm, 585 nm, and660 nm) when excited by light along certain wavelengths (e.g., about 470nm). The specific wavelengths at which a cell will fluoresce are acharacteristic of that cell and the wavelength(s) of the exciting light.The colorant also absorbs light at one or more predetermined wavelengthsas a function of the concentration of the colorant within the cell.Examples of acceptable colorants include the supravital dyes acridineorange and astrozone orange. The invention is not limited to supravitaldyes, however.

Now referring to FIG. 1, the analysis chamber 10 is defined by a firstpanel 12 having an interior surface 14, and a second panel 16 having aninterior surface 18. The panels 12, 16 are both sufficiently transparentto allow the transmission of light along predetermined wavelengths therethrough in an amount sufficient to perform the optical density analysisdescribed below. At least a portion of the panels 12, 16 are parallelwith one another, and within that portion the interior surfaces 14, 18are separated from one another by a height 20, which height may be knownor measurable. RBCs 22 are shown disposed within the chamber 10.

The present method can utilize a variety of different analysis chamberstypes having the aforesaid characteristics, and is not therefore limitedto any particular type of analysis chamber. An analysis chamber havingparallel panels 12, 16 simplifies the analysis and is thereforepreferred, but is not required for the present invention; e.g., achamber having one panel disposed at a known non-parallel angle relativeto the other panel could be used.

Now referring to FIGS. 2-5, an example of an acceptable chamber 10 isshown that includes a first panel 12, a second panel 16, and at leastthree separators 26 disposed between the panels 12, 16. The separators26 can be any structure that is disposable between the panels 12, 16,operable to space the panels 12, 16 apart from one another. Thedimension 28 of a separator 26 that extends between the panels 12, 16 isreferred to herein as the height 28 of the separator 26. The heights 28of the separators 26 typically do not equal one another exactly (e.g.,manufacturing tolerances), but are within commercially acceptabletolerance for spacing means used in similar analysis apparatus.Spherical beads are an example of an acceptable separator 26 and arecommercially available from, for example, Bangs Laboratories of Fishers,Ind., U.S.A.

In the chamber embodiment shown in FIG. 3, the separators 26 consist ofa material that has greater flexibility than one or both of the firstpanel 12 and the second panel 16. As can be seen in FIG. 3, the largerseparators 26 are compressed to the point where most separators 26 aretouching the interior surfaces of the panels 12, 16, thereby making thechamber height just slightly less than the mean separator 26 diameters.In the chamber embodiment shown in FIG. 4, the separators 26 consist ofa material that has less flexibility than one or both of the first panel12 and the second panel 16. In FIG. 4, the first panel 12 is formed froma material more flexible than the spherical separators 26 and the secondpanel 16, and will overlay the separators 26 in a tent-like fashion. Inthis embodiment, although small local regions of the chamber 10 maydeviate from the desired chamber height 20, the average height 20 of thechamber 10 will be very close to that of the mean separator 26 diameter.Analysis indicates that the mean chamber height 20 can be controlled toabout one percent (1%) or better at chamber heights of less than fourmicrons using this embodiment. Subject to the flexibilitycharacteristics described above (as well as other factors such as thedistribution density of the separators), the separators 26 and panels12, 16 can be made from a variety of materials, provided the panels 12,16 are sufficiently transparent. Transparent plastic films consisting ofacrylic or polystyrene are examples of acceptable panels 12, 16, andspherical beads made of polystyrene, polycarbonate, silicone, and thelike, are acceptable separators 26. A specific example of an acceptableseparator is spheres made of polystyrene that are commerciallyavailable, for example, from Thermo Scientific of Fremont, Calif.,U.S.A., catalogue no. 4204A, in four micron (4 μm) diameter. Referringto FIG. 5, the panel 12 that is to be vertically disposed above theother includes a plurality of ports 30 disposed at regular intervals(e.g., that act as air vents), and the panels 12, 16 are bonded togetherat points. In some embodiments, the bonding material 32 forms an outerchamber wall operable to laterally contain the sample 34 within theanalysis chamber 10. This example of an acceptable analysis chamber isdescribed in greater detail in U.S. Patent Application Publication Nos.2007/0243117, 2007/0087442, and U.S. Provisional Patent Application Nos.61/041,783, filed Apr. 2, 2008; and 61/110,341, filed Oct. 31, 2008, allof which are hereby incorporated by reference in their entirety.

Another example of an acceptable chamber 10 is disposed in a disposablecontainer 36 as shown in FIGS. 6 and 7. The chamber 10 is formed betweena first panel 12 and a second panel 16. Both the first panel 12 and thesecond panel 16 are transparent to allow light to pass through thechamber 10. At least a portion of the first panel 12 and the secondpanel 16 are parallel with one another, and within that portion theinterior surfaces 14, 18 are separated from one another by a height 20.This chamber 10 embodiment is described in greater detail in U.S. Pat.No. 6,723,290, which patent is hereby incorporated by reference in itsentirety. The analysis chambers shown in FIGS. 2-7, represent chambersthat are acceptable for use in the present method. The present method isnot, however, limited to these particular embodiments.

Some of the WBCs within the sample will likely contact both interiorsurfaces of the chamber panels and others will not. It is not arequirement that they contact the interior surfaces, and it is notnecessary to know the exact height of the chamber for purposes of thepresent invention. A chamber height of about two to six microns (2-6 μ)is acceptable for most animal species based on typical WBC sizes and thefact that WBCs can be deformed to some degree (e.g., partiallycompressed between the chamber interior surfaces). A chamber height 20of about three to five microns (3-5 μ) is particularly well suited foranalyzing human blood. An analysis of an animal species having WBCssubstantially larger or smaller than human WBCs can be performed in achamber respectively having a larger or smaller chamber height,respectively.

The analysis of the sample quiescently disposed within the chamber 10 isperformed using an analysis device that is operable to illuminate andimage at least a portion of the sample and perform an analysis on theimage. The image is produced in a manner that permits fluorescentemissions from, and the optical density of, the portion of the sample tobe determined on a per unit basis. The term “per unit basis” or “imageunit” means a defined incremental unit of which the image of the samplecan be dissected. A “pixel”, which is generally defined as the smallestelement of an image that can be individually processed within aparticular imaging system, is an example of an image unit, and an imageunit may also include a small number of pixels in a collective unit. Themagnification of an imaging device can also be described in linear terms(e.g., microns per pixel at the focal plane), where the linear dimensionis along a particular axis of an orthogonal grid applied to the image.The actual area of the sample captured by pixels of the sensor at thefocal plane is therefore a function of the magnification factor appliedby the imaging device. Hence, it is useful but not required to know themagnification of the imaging device. The volume associated with thatpixel is therefore the area of the image per pixel times the chamberheight. For example if the magnification was 0.5 microns per pixel, animage occupying 200 pixels would have an area of 50 square microns, anda volume of 50 square microns times the chamber height.

Now referring to FIG. 8, an example of an analysis device 44 that can beadapted for use with the present method includes a sample illuminator46, an image dissector 48, and a programmable analyzer 50. The sampleilluminator 46 includes a light source that selectively produces lightalong certain desired wavelengths. For example, LEDs that emit thedesired wavelengths (e.g., 420 nm, 440 nm, 470 nm, etc.) can be used.Alternatively, a light source that produces a broad wavelength range(e.g., approximately 400-670 nm) can be used, although in some instancessuch a light source may require filtering. The analysis device 44 mayinclude optics for manipulating the light. The sample illuminator 46includes a transmittance light source and an epi-illumination lightsource, each operable to illuminate some, or all, of the sample residingwithin the chamber 10. An example of an acceptable image dissector 48 isa charge couple device (CCD) that converts an image of the light passingthrough the sample into an electronic data format (i.e., a signal). Theprogrammable analyzer 50 includes a central processing unit (CPU) and isconnected to the sample illuminator 46 and image dissector 48. The CPUis adapted (e.g., programmed) to selectively perform the functionsnecessary to perform the present method. U.S. Pat. No. 6,866,823entitled “Apparatus for Analyzing Biologic Fluids” issued Mar. 15, 2005,which is hereby incorporated by reference in its entirety, disclosessuch an analysis device 44.

The analysis device is adapted to: 1) image at least a portion of thesample, and produce image signals indicative of fluorescent emissionsfrom the imaged sample and the optical density of the imaged sample on aper pixel basis; 2) determine a fluorescence value for one or moreconstituents of a first type and one or more constituents of a secondtype, all quiescently residing within the sample portion, using theimage signals; 3) determine an optical density value for each of theimaged first and second type constituents; and 4) identify the firsttype constituents and the second type constituents using the determinedfluorescence and optical density values.

Under the present method, a sample of substantially undiluted wholeblood is introduced into a chamber 10, and thereinafter residesquiescently as is described above. An anticoagulating agent and acolorant are admixed with the sample either prior to its introductioninto the chamber or upon introduction into the chamber. The colorant isabsorbed by the cells (e.g., WBCs and platelets) within the sample.Hereinafter, when referring to individual WBCs, the same procedureapplies to individual platelets, or other constituents within thesample. At least a portion of the sample quiescently residing within thechamber is illuminated by the analysis device 44, which transmits lightthrough the sample. Although it is not a requirement that the entiresample residing within the chamber be imaged, it is preferable sincedoing so typically provides a more complete analysis of the sample and aconcomitant increase in accuracy.

The sample is illuminated with wavelengths known to excite a fluorescentemission from the cells relating to the colorant absorbed by the WBCs.WBCs stained with acridine orange produce a fluorescent emission whenilluminated with violet light at a wavelength of about 470 nm Thespecific emissions depend upon the colorant used and the intracellularcomposition of the illuminated cell (e.g., interaction of the colorantwith the RNA and/or DNA of the cell creates the emissions). Some WBCshave fluorescent emissions that act as a fluorometric signature that isrelatively unique to that WBC and can therefore be used to identify thatWBC. Other WBCs have fluorescent emission signatures that cannot easilybe distinguished from one another. WBCs with those “shared” emissionsignatures may be grouped as being a first type WBC or a second typeWBC, but something further is required to distinguish the two WBC types.

At the same time the sample is illuminated to create a fluorescentemission (or sequentially thereafter), it is also illuminated along oneor more wavelengths that are absorbed by the colorant. WBCs stained withacridine orange, for example, absorb light at wavelengths of about 420nm due to the presence of the acridine orange. The amount of absorption,which can be described in terms of optical density (OD), is a functionof the concentration and local conditions (e.g., pH) of the colorantwithin the WBC. The propensity of a WBC to absorb a colorant, whenexposed to the same amount of colorant, varies between some WBC celltypes as a function of biological characteristics of the cell. Forexample, different biological characteristics within a WBC (e.g.,nuclear material, cytoplasm, etc.) will absorb dye in differentconcentrations. These different biological characteristics of each celltype, and the associated different concentrations of colorant absorbedby those characteristics, can be used to distinguish certain cell types.The OD of a cell, which is a function of the concentration of a colorantwithin the cell, can be used to distinguish and identify different celltypes. In some applications, the difference in OD between cells canprovide sufficient information to permit cell identification. In otherinstances, identification is accomplished using the fluorometricsignature and the OD of the cell.

To illustrate an example of the present invention, a substantiallyundiluted sample of blood is admixed with acridine orange and introducedwithin a chamber having two transparent panels. The sample residesquiescently and a plurality of WBCs within the sample contacts bothinterior surfaces of the chamber. The sample is illuminated at 470 nmand at 420 nm. The 470 nm illumination produces a fluorescent emission.The 420 nm illumination is absorbed by the colorant. Digital images ofthe illuminated sample are taken. A group of WBCs comprising neutrophilsand eosinophils are identified within the entire WBC population presentwithin the imaged sample, and that group is “separated” within theimage; e.g., by filtering the image so that only the group can be seen.The neutrophils and the eosinophils are identified because each of theseWBC types produces a signature fluorescence pattern upon excitation,consisting of a significant red cytoplasmic fluorescence and a greennuclear fluorescence. The fluorescent emissions of the neutrophils andthe eosinophils within the group are, however, sufficiently similar toone another that it is difficult to distinguish the two types of WBCs.

To distinguish between the two types of WBCs within the group, theoptical density of the separated WBCs are compared. On average, theconcentration of the acridine orange absorbed within the eosinophils isgreater than the concentration of the acridine orange absorbed withinthe neutrophils, although the fluorescence may be the same. This isbecause the fluorescence of the colorant within the eosinophils isquenched relative to that within the neutrophils because of the uniqueattributes of the cellular contents of the eosinophil. The two differenttypes of WBCs can be distinguished as separate subgroups, for example,by using a predetermined OD cutoff value; e.g., those cells within theseparated group having an OD greater than the cutoff value are labeledas eosinophils, and those cells having an OD that is less than thecutoff value are labeled as neutrophils.

Alternatively, the two types of WBCs can be distinguished by comparingtheir measured OD to empirically derived OD values stored within theanalysis device; e.g., in a look up table, etc.

Still further, the two WBC subgroups can be distinguished from oneanother by determining the ratio of cytoplasmic fluorescence tocytoplasmic OD (fluorescence/OD) on an individual cell basis. To createthe ratio, the fluorescent emission values and the optical densityvalues on a per pixel basis for a particular cell can be determined andaveraged, and the average values can be used within the ratio. The ratiocan be determined using alternative methods such as determining theratio on a per pixel basis and averaging the per pixel ratios. The ratioof fluorescence to OD quantitatively expresses the quenching of thestain's fluorescence within a particular cell. Cells having a lowerratio show “quenching” of the fluorescent signal. The ratios of all thecells within the separated group can be statistically evaluated todetermine a point of separation between two populations. The cellsstatistically falling below the point of separation are the eosinophilsbecause the ratio of fluorescence to OD is lower than the ratioassociated with the population of neutrophils. Similarly, the cellsstatistically above the point of separation are the neutrophils becausethe ratio of fluorescence to OD is higher than the ratio of thepopulation of eosinophils.

In a further embodiment of the above fluorescence/OD ratio analysis, theratios can be determined using only above average OD and fluorescentemission values (or OD values and fluorescent emission values withinpercentage that is greater the 50%) from the cells under examination. Toexplain, the concentration of colorant in a particular cell exposed tothe colorant may be less in a first region (e.g., nuclear region) thanit is in a second region (e.g., cytoplasm region). Consequently the ODof the second region of the cell (e.g. cytoplasm) will be greater thanthe OD of the first region (e.g., nuclear) of the cell. In similarfashion, the fluorescent emissions from a particular region of a cellmay be greater than the emissions from another region. Selectively usinga portion of the fluorescent emission/OD values, which values representgreater emission intensity or OD, results in an improved noise to signalratio that facilitates the analysis. This aspect takes advantage of thefact that colorants typically preferentially distribute, for example,within the granules within the cytoplasm of the cells.

In a further embodiment of the present invention, the cells within thesample can be distinguished from one another by “bleaching” the cellsadmixed with the colorant with a constant emission of light at awavelength (e.g., 470 nm) that excites a fluorescent emission, andsensing the magnitude of the emitted light at discrete points in timewithin a period of time. The average rate at which fluorescent emissionsdecrease in intensity from a particular cell type is constant for thatcell type, but the average rates vary as between types of cells.Consequently, the decremental rate of intensity emission can be used todistinguish cell types. For example, FIG. 9 illustrates the rate ofdecay of green fluorescence emission for individual neutrophils exposedto photobleaching 52, 54, 56, and the average rate of decay for thoseneutrophils 58. FIG. 10 illustrates the rate of decay of greenfluorescence emission for individual lymphocytes exposed to the samephotobleaching 60, 62, and for the average 64 of those lymphocytes. Thecurves shown in FIGS. 9 and 10 clearly show different decay rates forthe different cell types, and that the different rates of decay can beused to identify the type of cell being analyzed. The decremental rateof emission can also be used to distinguish cells by othercharacteristics such as age; e.g., cells of a certain type but differentin age will have characteristic decremental rates of emission that canbe used to distinguish the various different age groups.

Although this invention has been shown and described with respect to thedetailed embodiments thereof, it will be understood by those skilled inthe art that various changes in form and detail may be made withoutdeparting from the spirit and scope of the invention.

What is claimed is:
 1. A method for analyzing a quiescently residingbiologic fluid sample, which sample has at least one first constituentand at least one second constituent, and which second constituent is adifferent type than that of the first constituent, and which sample ismixed with a colorant, the method comprising: using a processor toexecute instructions stored in a memory device, which instructions causethe processor to: control a sample illuminator to illuminate at least aportion of the sample containing the first constituent and the secondconstituent with light at one or more wavelengths that cause thecolorant to fluoresce, and with light at one or more wavelengths thatare absorbed by the colorant; control an imaging device to image the atleast a portion of the sample, including producing first image signalsindicative of fluorescent emissions from the colorant disposed withinthe first constituent and second image signals from the colorantdisposed within the second constituent, and including producing thirdimage signals indicative of the optical density of at least one regionwithin the first constituent and producing fourth image signalsindicative of the optical density of at least one region within thesecond constituent; determine at least one value representative offluorescent emissions using the first image signals and at least onevalue representative of fluorescent emissions using the second imagesignals; determine at least one value representative of optical densityassociated with the first constituent using the third image signals andat least one value representative of optical density associated with thesecond constituent using the fourth image signals; and identify thefirst constituent and the second constituent using the determinedrepresentative fluorescent emission values and the determinedrepresentative optical density values.
 2. The method of claim 1, whereinthe first constituent and the second constituent are selected from thegroup consisting of eosinophils, neutrophils, and basophils.
 3. Themethod of claim 1, wherein the first and second constituents areselected from the group consisting of white blood cells andparticulates.
 4. The method of claim 3, wherein the first and secondconstituents are each types of white blood cells.
 5. An apparatus foranalyzing a quiescently residing biologic fluid sample, which sample hasat least one first constituent and at least one second constituent, andwhich second constituent is a different type than that of the firstconstituent, and which sample is mixed with a colorant, comprising: asample illuminator; an imaging device; a processor that executesinstructions; a memory device that stores logic that, when executed bythe processor, causes the apparatus to perform at least the following:control the sample illuminator to illuminate at least a portion of thesample containing the first constituent and the second constituent withlight at one or more wavelengths that cause the colorant to fluoresce,and with light at one or more wavelengths that are absorbed by thecolorant; control the imaging device to image the at least a portion ofthe sample, including producing first image signals indicative offluorescent emissions from the colorant disposed within the firstconstituent and second image signals from the colorant disposed withinthe second constituent, and including producing third image signalsindicative of the optical density of at least one region within thefirst constituent and producing fourth image signals indicative of theoptical density of at least one region within the second constituent;determine at least one value representative of fluorescent emissionsusing the first image signals and at least one value representative offluorescent emissions using the second image signals; determine at leastone value representative of optical density associated with the firstconstituent using the third image signals and at least one valuerepresentative of optical density associated with the second constituentusing the fourth image signals; and identify the first constituent andthe second constituent using the determined representative fluorescentemission values and the determined representative optical densityvalues.